Elastin stabilization of connective tissue

ABSTRACT

A method and product are provided for the treatment of connective tissue weakened due to destruction of tissue architecture, and in particular due to elastin degradation. The treatment agents employ certain unique properties of phenolic compounds to develop a protocol for reducing elastin degradation, such as that occurring during aneurysm formation in vasculature. According to the invention, elastin can be stabilized in vivo and destruction of connective tissue, such as that leading to life-threatening aneurysms in vasculature, can be tempered or halted all together. The treatment agents can be delivered or administered acutely or chronically according to various delivery methods, including sustained release methods incorporating perivascular or endovascular patches, use of microsphere carriers, hydrogels, or osmotic pumps.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/827,121 filed Jul. 10, 2007, now U.S. Pat. No. 7,713,543entitled “Elastin Stabilization of Connective Tissue”, incorporatedherein by reference, which is a continuation of U.S. patent applicationSer. No. 11/387,454, filed Mar. 23, 2006, now U.S. Pat. No. 7,252,834,incorporated herein by reference, which has a priority date based onProvisional Application Ser. No. 60/674,631, which was filed on Apr. 25,2005.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

The United States Government may have rights to this invention pursuantto National Institutes of Health Grant No. HL-61652.

BACKGROUND OF THE INVENTION

One of the most common results of the degradation of vasculature isaneurysm. By definition, the term “aneurysm” is simply an abnormalwidening or ballooning at the wall of a blood vessel. This condition canbe devastating due to the potential for rupture or dissection that canlead to massive bleeding, stroke, or hemorrhagic shock, and can be fatalin an estimated 80% of cases. Aneurysms can be caused by any of a largeclass of degenerative diseases and pathologies including atheroscleroticdisease, defects in arterial components, genetic susceptibilities, andhigh blood pressure, among others, and can develop silently over aperiod of years. The hallmarks of aneurysms include enzymaticdegradation of vascular structural proteins such as elastin,inflammatory infiltrates, calcification, and eventual overalldestruction of the vascular architecture. For example, FIG. 1graphically illustrates the difference in elastin content between ahealthy aorta and an aneurysmal aorta. As can be seen, elastin contentof the damaged structure is 70% less than that of the healthy structure.

Current methods of treatment for diagnosed aneurysms are limited toinvasive surgical techniques. After initial diagnosis of a smallaneurysm, the most common medical approach is to follow up thedevelopment of the aneurysm and after reaching a pre-determined size(e.g., about 5 cm in diameter), surgical treatment is applied. Currentsurgical treatments are limited to either an endovascular stent graftrepair or optionally complete replacement of the diseased vessel with avascular graft. While such surgical treatments can save lives andimprove quality of life for those suffering aneurysm, dangers beyondthose of the surgery itself still exist for the patient due to possiblepost-surgery complications (e.g., neurological injuries, bleeding, orstroke) as well as device-related complications (e.g., thrombosis,leakage, or failure). Moreover, depending upon the location of theaneurysm, the danger of an invasive surgical procedure may outweigh thepossible benefits of the procedure, for instance in the case of ananeurysm deep in the brain, leaving the sufferer with very little in theway of treatment options. Moreover, surgical treatments may not alwaysprovide a permanent solution, as vascular grafts can loosen and dislodgeshould the aneurysm progress following the corrective surgery.

Aneurysm is not the only condition for which enzymatic degradation ofstructural proteins is a hallmark. Other conditions in which structuralprotein degradation appears to play a key role include Marfan syndrome,supravalvular aortic stenosis, and chronic obstructive pulmonary disease(COPD). For those afflicted, such conditions lead to, at the very least,a lowered quality of life and often, premature death.

Phenolic compounds are a diverse group of materials that have beenrecognized for use in a wide variety of applications. For instance, theynaturally occur in many plants, and are often a component of the humandiet. Phenolic compounds have been examined in depth for efficacy asfree radical scavengers and neutralizers, for instance in topical skinapplications and in food supplements. Phenolic compounds are alsobelieved to prevent cross-linking of cell membranes found in certaininflammatory conditions and are believed to affect the expressions ofspecific genes due to their modulation of free radicals and otheroxidative species (see, e.g., U.S. Pat. No. 6,437,004 to Perricone).

What are needed in the art are treatment protocols and compositions forstabilization of the organs and tissues affected by degenerativeconditions such as aneurysm. In particular, treatment protocolsutilizing phenolic compounds could provide a safe, less invasive routefor the stabilization of the structural architecture in order to tempergrowth and/or development of such conditions.

SUMMARY OF THE INVENTION

In one embodiment, the present invention is directed to a method forstabilizing connective tissue containing elastin. For instance, themethod can include applying a phenolic compound directly to connectivetissue. Phenolic compounds for use in the disclosed methods include ahydrophobic core and at least one phenolic group joined to thehydrophobic core.

In one preferred embodiment, the connective tissue targeted by theprotocol can be a component of a blood vessel. For instance, the methodcan be utilized to treat arteries, and in one particular embodiment, theaorta.

Methods of the invention can include providing a phenolic compound in asuitable drug delivery vehicle such as, for example, a sustained releasedrug delivery vehicle. In one embodiment, a drug delivery vehicle, e.g.,a microencapsulation, a hydrogel, an implantable device such as a stent,a patch, a vascular graft, or the like, can be located adjacent to theconnective tissue for direct delivery of the phenolic compound to thetissue.

In other embodiments, other delivery methods and devices may be used.For instance, a composition including a phenolic compound and aparenterally acceptable carrier can be injected directly into theconnective tissue. In yet another embodiment, the disclosed compoundscan be delivered to the connective tissue of a blood vessel wall viaintravenous delivery.

Methods and compositions described herein can be beneficially utilizedin one embodiment for in vivo therapeutic or prophylactic treatments,and in one particular embodiment, for treatment of aneurysmal bloodvessels.

The present invention is also directed to compositions that can be usedin the methods. For instance, compositions of the invention can includebetween about 0.0001 w/v % and about 10 w/v % of a phenolic compound anda parenterally acceptable carrier. In general, the composition can havea pH between about 4 and about 9, for instance between about 5.5 andabout 7.

In one preferred embodiment, the phenolic compound can be a tannin or atannin derivative. For instance, the phenolic compound can bepentagalloylglucose.

BRIEF DESCRIPTION OF THE FIGURES

A full and enabling disclosure of the present invention, including thebest mode thereof, to one of ordinary skill in the art, is set forthmore particularly in the remainder of the specification, includingreference to the accompanying drawings in which:

FIG. 1 is a graphical representation of the difference in elastincontent of a healthy aorta and an aneurysmal aorta in an animal model;

FIG. 2 illustrates the chemical structure of tannic acid, withpentagalloylglucose highlighted by the circle;

FIG. 3 schematically illustrates exemplary delivery methods for phenoliccompounds according to certain embodiments of the present inventionincluding perivascular delivery (FIG. 3A) and endovascular delivery(FIG. 3B);

FIGS. 4A-4D show stained aortic tissue sections described in the examplesection illustrating stabilization of elastin of the connective tissuein the presence of elastase;

FIG. 5A illustrates the procedures used for examination of the openingangle of a section of porcine aorta and as described in the examplesection;

FIG. 5B graphically illustrates the results of the determination of theopening angle of the sections of porcine aorta;

FIG. 5C includes photographs of the porcine aorta sections showingrecoil evaluation of treated and control tissue samples;

FIGS. 6A-6E are digital photographs showing results for the live/deadassay of treated tissue samples as described in the example section;

FIG. 7 is a graphic representation showing the kinetics of the bindingof tannic acid to pure aortic elastin;

FIG. 8 graphically illustrates the increase in diameter upon injury ofrat aortas (no treatment) and illustrates the significant reduction inaneurysm for aortas treated with a phenolic compound as hereindescribed;

FIG. 9 illustrates the percentage increase in diameter for the controlaortas and the treated aortas of FIG. 8;

FIG. 10A graphically illustrates the elastin content of the controlaortas and the treated aortas of FIG. 8;

FIGS. 10B and 10C illustrate a control aorta and a treated aortafollowing Verhoeff van Giesson staining; and

FIG. 11 compares the results following extraction of phenolic compoundsfrom treated tissue immediately following treatment as herein describedin situ and 28 days in vivo following treatment.

DETAILED DESCRIPTION OF THE INVENTION

Reference now will be made in detail to embodiments of the invention,one or more examples of which are set forth below. Each example isprovided by way of explanation of the invention, not limitation of theinvention. In fact, it will be apparent to those skilled in the art thatvarious modifications and variations can be made in the presentinvention without departing from the scope or spirit of the invention.For instance, features illustrated or described as part of oneembodiment, can be used on another embodiment to yield a still furtherembodiment.

The present invention is generally directed to methods and phenoliccompounds that can be beneficially utilized for stabilization ofconnective tissue. In particular, the presently disclosed methods andcompounds can stabilize the elastin component of connective tissue, andthereby prevent the degradation of the structural architecture of theconnective tissue. For instance, the disclosed methods can be utilizedto prevent the structural breakdown of organs incorporating the targetedconnective tissue, e.g., blood vessels. In one particular embodiment,the materials and methods disclosed herein can be utilized in thetreatment of chronic connective tissue degenerative conditions. Forinstance, the invention can be directed to in vivo treatment methods andcompositions. Connective tissue targeted by the process can bestabilized so as to be less susceptible to protein degradation that canbe brought about due to any of a variety of mechanisms and/or conditionsincluding, for example, those associated with aneurysm, atheroscleroticdisease, genetic susceptibilities, blunt force injury, Marfan'ssyndrome, and the like.

Connective tissue is the framework upon which the other types of tissue,i.e., epithelial, muscle, and nervous tissues, are supported. Connectivetissue generally includes individual cells not directly attached to oneanother and held within the extracellular matrix. The extracellularmatrix, in turn, includes the ground substance (e.g., the minerals ofbone, the plasma of blood, etc.) and the fibrous component includingcollagen fibers and elastin fibers. Connective tissue can assume widelydivergent architectures, ranging from blood, in which the fibrouscomponent is absent and the ground substance is fluid, to denseconnective tissue, which includes a relatively high proportion ofextracellular fibers (e.g., collagen) and may contain little of theother connective tissue components. There are many specialized types ofconnective tissue, one example being elastic tissue, in which elasticfibers are the major component of the tissue and the amount of factorscommonly found in other types of connective tissue, such as collagen andproteoglycans, may be minimal.

The compounds and processes disclosed herein are directed tostabilization of the elastin component of connective tissue, and in oneparticular embodiment, stabilization of the elastin component ofvasculature. It should be understood that while the invention isdirected in one particular embodiment to the stabilization of bloodvessels susceptible to the formation of aneurysms, in other embodimentsother organs, other diseases and/or other conditions can be treated. Inparticular, the disclosed treatment agents and treatment protocols areapplicable to any animal or human connective tissue that include anelastin component.

Elastin is the protein constituent of connective tissue responsible forthe elasticity and recoil of the tissue. Moreover, elastin is quiteabundant in connective tissue. In fact, elastin is the most abundantextracellular matrix protein found in the aortic wall. Elastinpolypeptide chains are naturally cross-linked together to formrubber-like, elastic fibers. Unlike collagen, elastin molecules canuncoil into a more extended conformation when the fiber is stretched andwill recoil spontaneously as soon as the stretching force is relaxed.Elastin degeneration in connective tissue pathology is generally causedby enzymes including elastase enzymes and matrix metalloproteinase (MMP)enzymes that can be secreted by vascular cells as well as byinfiltrating inflammatory cells. While many aspects of the methods andschemes of various enzymes leading to elastin degradation remainunknown, in general, it is believed that most enzymes attack and bindthe protein at a site away from the crosslinks.

According to the present invention, degradation of connective tissue canbe prevented or slowed through the stabilization of the elastincomponent of the tissue with a phenolic compound. In particular, it isbelieved that any of a number of natural and synthetic phenoliccompounds can bind elastin and thereby protect elastin from degradation,for instance due to the action of elastin degrading enzymes.Accordingly, in one embodiment, the present invention is directed tomethods and compounds that can inhibit enzyme-catalyzed degradation ofelastin, and in particular elastase and/or MMP catalyzed degradation ofelastin.

Phenolic compounds encompassed by the present invention include anycompound that includes at least one phenolic group bound to ahydrophobic core. While not wishing to be bound by any particulartheory, it is believed that interaction between the phenolic compoundand elastin proteins include aspects involving both the hydroxyl groupas well as the hydrophobic core of the molecules. In particular, it isbelieved that phenolic compounds can stabilize elastin proteins throughboth steric means and bond formation and thereby protect sites on theprotein susceptible to enzyme-mediated (e.g., elastase or MMP-mediated)cleavage. Specifically, it is believed that hydroxyl groups of aphenolic compound can bind elastin multivalently, for instance viahydrogen bond formation with amino acid residues such as polar aminoacid residues including methionine, glycine and proline, such thatmultiple proteins can interact with a single molecule to create athree-dimensional cross-link structure involving multiple elastinmolecules. Moreover, in certain embodiments, the phenolic compounds ofthe present invention can include one or more double bonds, with whichthe phenolic compounds can covalently bind to the elastin, forming aneven stronger and more permanent protective association between thephenolic compound and the elastin of the connective tissue. In addition,the large hydrophobic regions of the elastin protein, which are believedto contain sites susceptible to elastase-mediated cleavage, are alsobelieved to contain sites of association between the hydrophobic core ofthe phenolic compound and the protein. Thus, the association between thephenolic compound and the protein molecules are believed to protectspecific binding sites on the protein targeted by enzymes through theassociation of the protein with the hydrophobic core and can alsosterically hinder the degradation of the protein through the developmentof the large three dimensional cross-link structure.

Phenolic compounds encompassed by the present invention includematerials including a hydrophobic core and one or more phenol groupsextending from the hydrophobic portion of the molecule. For instance,exemplary phenolic compounds of the invention can include, but are notlimited to, flavonoids and their derivatives (e.g., anthocyanins,quercetin), flavolignans, phenolic rhizomes, flavan-3-ols including(+)-catechin and (−)-epicatechin, other tannins and derivatives thereof(such as tannic acid, pentagalloylglucose, nobotanin, epigallocatechingallate, and gallotannins), ellagic acid, procyanidins, and the like.

Phenolic compounds of the invention include synthetic and naturalphenolic compounds. For example, natural phenolic compounds can includethose found in extracts from natural plant-based sources such asextracts of olive oil (e.g., hydroxytyrosol (3,4-dihydroxyphenylethanol)and oleuropein, extracts of cocoa bean that can contain epicatechin andanalogous compounds, extracts of Camellia including C. senensis (greentea) and C. assaimic, extracts of licorice, sea whip, aloe vera,chamomile, and the like.

In one preferred embodiment, the phenolic compounds of the invention canbe tannins and derivatives thereof. Tannins can be found in many plantspecies. For example, the tea plant (Camellia sinensis) has a naturallyhigh tannin content. Green tea leaves are a major plant source oftannins, as they not only contain the tannic and gallic acid groups, butalso prodelphinidin, a proanthocyanidin. Tannins are also found in wine,particularly red wine as well as in grape skins and seeds. Pomegranatesalso contain a diverse array of tannins, particularly hydrolysabletannins.

Tannic acid, the structure of which is illustrated in FIG. 2, is acommon naturally derived tannin. Tannic acid, as a cross-linking agent,is similar in many properties to that of many fixatives often used inthe preparation and formation of xenograft or allograft tissue implants,for instance glutaraldehyde fixatives. Moreover, tannic acid caninteract with other connective tissue components as well as elastin, andthus can stabilize additional components of the targeted connectivetissue in the disclosed processes, in addition to the elastin component.For instance, tannic acid is capable of cross-linking glycosaminoglycanpolysaccharides as well as other connective tissue components.

In one embodiment, the present invention is directed to utilization ofthe disclosed agents for stabilization of connective tissue in vivo.Accordingly, in such embodiments, biocompatibility and cytotoxicity ofthe agents can be of importance in preparation of therapeutics includingthe disclosed compounds. At one time, tannic acid-containingpreparations were suspected of causing hepatoxicity. This toxicity hassince been primarily attributed to poor purity of the preparations andthe inclusion of toxic gallic acid residues in the compositions.Accordingly, in one embodiment, the present invention is directed tocompositions including high purity tannic acid, with little or no freegallic acid residue included in the compositions. For example, in oneembodiment, the compositions of the present invention can include lessthan about 5% free gallic acid residue in the preparation. In oneembodiment, the compositions of the present invention can includebetween about 1% and about 5% free gallic acid residue in thecomposition.

In one preferred embodiment of the present invention, compositions aredisclosed comprising an effective amount of pentagalloylglucose (PGG).PGG, which is the portion of a tannic acid molecule enclosed in thecircle in FIG. 2, includes the hydrophobic core of tannic acid as wellas multiple phenolic hydroxy groups, but does not posses the outergallic acid residues and the hydrolyzable ester bonds associated withtannic acid. Thus, the possibility of release of free gallic acidresidues over the course of a long-term application process can beprevented in one embodiment of the invention through utilization of acompound having no gallic acid residues, such as PGG, as the selectedagent.

In general, the phenolic compounds described herein can be provided as abiocompatible composition. For instance, compositions disclosed hereincan include one or more phenolic compounds in a concentration that canvary over a wide range, with a preferred concentration generallydepending on the particular application, the delivery site targeted bythe phenolic compound and the mode that will be used in the deliveryprocess. For example, in one embodiment, a composition of the inventioncan include one or more phenolic compounds at a concentration from about0.0001% to about 10%. (Unless otherwise noted, all concentrationsreported herein are weight/volume percentages.) It should be noted,however, that while these exemplary concentrations are effective incertain embodiments, the invention encompasses compositions comprising awider range of phenolic compound concentrations. For example, actualconcentrations used may be influenced by the organ targeted by theprocedure, size of the targeted area, desired incubation time, andpreferred pH, in addition to delivery mode, as mentioned above. In oneembodiment of the present invention, the disclosed compositions caninclude concentrations of a phenolic compound ranging from about 0.1% toabout 1%.

In one embodiment, the phenolic compounds can be provided inpharmaceutically acceptable formulations using formulation methods knownto those of ordinary skill in the art. These formulations can generallybe administered by standard routes. For example, the formulations may beadministered in one embodiment directly to connective tissue, forinstance through exposure of the connective tissue and directapplication thereto, or via direct injection of the formulation to thetargeted connective tissue. In other embodiments, however, theformulations may be administered indirectly to the targeted tissue.

The formulations can be delivered intravenously in a systemic deliveryprotocol. For example, osmotic mini-pumps may be used to providecontrolled delivery of high concentrations of the treatment agentsthrough cannulae to the site of interest, such as directly into atargeted blood vessel. In situ polymerizable hydrogels, as are generallyknown to those of skill in the art, and discussed further below, areanother example of a delivery vehicle that can be utilized in a deliveryprotocol, for instance in an intravenous delivery directly to targetedcannulae. Once delivered to the targeted blood vessel by any suitablemethod as is accepted in the art, the phenolic compound can penetratethe vessel wall and stabilize the connective tissue of the vessel. Forinstance, when delivered to the connective tissue from the lumen of ablood vessel, the phenolic compounds disclosed herein penetrate theendothelium of the vessel wall to contact the elastin of the connectivetissue and stabilize the structure architecture.

Compositions of the present invention can include additional agents, inaddition to the phenolic compounds. Such agents can be active agents,providing direct benefit to the tissue in addition to the stabilizationprovided by the phenolic compound, or may be supporting agents,improving delivery, compatibility, or reactivity of other agents in thecomposition. For example, in one embodiment, the compositions caninclude glutaraldehyde. Glutaraldehyde, when targeted to connectivetissue, can form covalent cross-links between free amines in proteins inorder to further stabilize the tissue. If desired, the composition canincorporate a gallic acid scavenger, for example ascorbic acid orglutathione, so as to prevent the release of free gallic acid residues.

The phenolic compounds can be combined with any of a number of possiblelipid-lowering medications so as to prevent the development of calcifiedlipid deposits or arteriosclerosis plaques that can often be found inconjunction with aneurysm formation.

A phenolic composition of the invention can include one or more buffersas are generally known in the art. For example, a composition includingone or more phenolic compounds and having a pH from about 4.0 to about9.0 may be formulated with inclusion of a biocompatible buffer such asdistilled water, saline, phosphate buffers, borate buffers, HEPES,PIPES, and MOPSO. In one embodiment, a composition of the invention maybe formulated to have a pH of between about 5.5 and about 7.4.

Compositions for parenteral delivery, e.g., via injection, can includepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions as well as sterile powders forreconstitution into sterile injectable solutions or dispersions justprior to use. Examples of suitable aqueous and nonaqueous carriers,diluents, solvents or vehicles include water, ethanol, polyols (e.g.,glycerol, propylene glycol, polyethylene glycol and the like),carboxymethylcellulose and suitable mixtures thereof, vegetable oils(e.g., olive oil) and injectable organic esters such as ethyl oleate. Inaddition, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like that can enhance the effectiveness of the phenoliccompound. Proper fluidity may be maintained, for example, by the use ofcoating materials such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions and by the use of surfactants.These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents.

Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It may also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like.

In one embodiment, the compositions can include pharmaceuticallyacceptable salts of the components therein, e.g., those that may bederived from inorganic or organic acids. Pharmaceutically acceptablesalts are well known in the art. For example, S. M. Berge, et al.describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences (1977) 66:1 et seq., which is incorporatedherein by reference. Pharmaceutically acceptable salts include the acidaddition salts that are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, tartaric, mandelic and the like. Salts formed with free carboxylgroups can also be derived from inorganic bases such as, for example,sodium, potassium, ammonium, calcium or ferric hydroxides, and suchorganic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine and the like. The salts may be prepared in situduring the final isolation and purification of the compounds of theinvention or separately via reaction of a free base function with asuitable organic acid. Representative acid addition salts include, butare not limited to acetate, adipate, alginate, citrate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,camphorsulfonate, digluconate, glycerophosphate, hemisulfate,heptonoate, hexanoate, fumarate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxymethanesulfonate (isethionate), lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate and undecanoate. Also, the basicnitrogen-containing groups can be quaternized with such agents as loweralkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl,and diamyl sulfates; long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides; arylalkyl halides likebenzyl and phenethyl bromides and others. Water or oil-soluble ordispersible products are thereby obtained. Examples of acids which maybe employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, hydrobromic acid,sulphuric acid and phosphoric acid and such organic acids as oxalicacid, maleic acid, succinic acid and citric acid.

In one embodiment the method can include use of timed release orsustained release delivery systems as are generally known in the art.Such systems can be desirable, for instance, in situations where longterm delivery of the agents to a particular organ or vascular locationis desired. According to this particular embodiment, a sustained-releasematrix can include a matrix made of materials, usually polymers, whichare degradable by enzymatic or acid/base hydrolysis or by dissolution.Once located at or near the target tissue, e.g., inserted into the body,for instance in the form of a patch or a stent such as those furtherdescribed below, such a matrix can be acted upon by enzymes and bodyfluids. The sustained-release matrix can be chosen from biocompatiblematerials such as liposomes, polylactides (polylactic acid),polyglycolide (polymer of glycolic acid), polylactide co-glycolide(co-polymers of lactic acid and glycolic acid) polyanhydrides,poly(ortho)esters, polyproteins, hyaluronic acid, collagen, chondroitinsulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides,nucleic acids, polyamino acids, amino acids such as phenylalanine,tyrosine, isoleucine, polynucleotides, polyvinyl propylene,polyvinylpyrrolidone and silicone. Possible biodegradable polymers andtheir use are described, for example, in detail in Brem et al. (1991, J.Neurosurg. 74:441-6), which is hereby incorporated by reference in itsentirety.

When an effective amount of the agents of the present invention areadministered by intravenous or subcutaneous injection, the compositionscan generally be in the form of a pyrogen-free, parenterally acceptableaqueous solution. The preparation of such parenterally acceptablesolutions, having due regard to pH, isotonicity, stability, and thelike, is within the skill in the art. A preferred pharmaceuticalcomposition for intravenous, cutaneous, or subcutaneous injection cancontain, in addition to the phenolic compound of the present invention,an isotonic vehicle such as Sodium Chloride Injection, Ringer'sInjection, Dextrose Injection, Dextrose and Sodium Chloride Injection,Lactated Ringer's Injection, or other vehicle as known in the art. Thetreatment composition of the present invention may also containstabilizers, preservatives, antioxidants, or other additives known tothose of skill in the art.

The dosage of the disclosed treatment agents of the present inventioncan depend on the disease state or particular condition being treatedand other clinical factors such as weight and condition of the human oranimal and the route of administration of the compound. The disclosedtreatment agents can be administered between several times per day to asingle treatment protocol. Optionally, treatment agents could bedelivered according to the disclosed process either acutely, during aone-time intervention, or chronically, for instance using multipleadministrations or optionally a single administration of a timed orsustained releases system. It is to be understood that the presentinvention has application for both human and veterinary use. The methodsof the present invention contemplate single as well as multipleadministrations, given either simultaneously or over an extended periodof time. In addition, the disclosed treatment agents can be administeredin conjunction with other forms of therapy, e.g., surgical endovascularstent graft repair or replacement of an excessively damaged area ofvasculature.

In one embodiment, a composition including one or more phenoliccompounds can be targeted to a specific site, such as to a diagnosedaneurysm in vivo, using a minimally invasive procedure to providelong-term delivery of the treatment agent locally from a biocompatibleimplantable device. For example, FIG. 3 schematically illustratespossible perivascular (FIG. 3A) and endovascular (FIG. 3B) routes ofdelivery, either of which could be utilized for local targeted deliveryof the disclosed treatment agents, for instance to a diagnosed aneurysm.

Perivascular delivery technologies suitable for use in the presentinvention are generally known to those of skill in the art, and thusneed not be explained at length herein. For instance, exemplary knownperivascular drug delivery technologies include those described by Chen,et al. (U.S. patent Application Publication No. 2005/0079202) and Nathan(U.S. Patent Application Publication No. 2003/0228364), both of whichare incorporated herein by reference. These exemplary perivasculardelivery systems include a polymeric delivery vehicle that can beinjected or directly placed, for instance via surgery, at a particularlocation so as to provide controlled release of the phenolic compoundencapsulated or otherwise loaded therein over a period of time.

A phenolic compound of the invention can be loaded in a drug deliveryvehicle via encapsulation, coating, infusion, or any other loadingmechanism as is known in the art.

Prolonged absorption of an injectable pharmaceutical form may be broughtabout by the inclusion of agents, such as aluminum monostearate andgelatin, which can delay absorption. For example, injectable depot formscan be made by forming microencapsule matrices including the phenoliccompound loaded in the matrix formed of biodegradable polymers such aspolylactide-polyglycolide, poly(orthoesters) and poly(anhydrides).Depending upon the ratio of therapeutic agent to polymer and the natureof the particular polymer employed, the rate of drug release can becontrolled. Depot injectable formulations can also be prepared byentrapping the therapeutic agents in liposomes or microemulsions whichare compatible with body tissues. The injectable formulations may besterilized, for example, by filtration through a bacterial-retainingfilter or by incorporating sterilizing agents in the form of sterilesolid compositions which can be dissolved or dispersed in sterile wateror other sterile injectable media just prior to use.

Many endovascular drug delivery methods are likewise known in the art.For example, DiCarlo, et al. (U.S. Pat. No. 6,929,626, incorporatedherein by reference) describe an intraluminally placeable tubular devicethat can be located within the lumen of a blood vessel and coated orotherwise loaded with a drug, e.g., the phenolic compounds describedherein. The tubular member includes yarns interconnected in a patterndefining opposed interior and exterior textile surfaces. At least one ofthe textile surfaces is the body fluid-contacting luminal surface or thebody lumen-contacting exterior surface.

Wu, et al. (U.S. Pat. No. 6,979,347, incorporated herein by reference)describe an apparatus and associated method for delivering a therapeuticsubstance such as the phenolic compounds of the present invention, to avascular lumen. Specifically, an implantable prosthesis, such as astent, can be utilized that has grooves or trenches formed thereon. Thegrooves are formed on specific regions of the stent struts to increasethe flexibility of the stent. The grooves also provide a location forcarrying the phenolic compound for delivery from the device followingimplantation. For example, the phenolic compound, or a compositionthereof, can be deposited directly in to the grooves using conventionalspray or modified dip techniques.

In another embodiment, the disclosed agents can be targeted toconnective tissue by use of a hydrogel delivery vehicle. Hydrogels areherein defined to include polymeric matrices that can be highly hydratedwhile maintaining structural stability. Suitable hydrogel matrices caninclude un-crosslinked and crosslinked hydrogels. In addition,crosslinked hydrogel delivery vehicles of the invention can optionallyinclude hydrolyzable portions, such that the matrix can be degradablewhen utilized in an aqueous environment, e.g., in vivo. For example, thedelivery vehicle can include a cross-linked hydrogel including ahydrolyzable cross-linking agent, such as polylactic acid, and can bedegradable in vivo.

Hydrogel delivery vehicles of the present invention can include naturalpolymers such as glycosaminoglycans, polysaccharides, proteins, and thelike, as well as synthetic polymers, as are generally known in the art.A non-limiting list of hydrophilic polymeric materials that can beutilized in forming hydrogels of the present invention can includedextran, hyaluronic acid, chitin, heparin, collagen, elastin, keratin,albumin, polymers and copolymers of lactic acid, glycolic acid,carboxymethyl cellulose, polyacrylates, polymethacrylates, epoxides,silicones, polyols such as polypropylene glycol, polyvinyl alcohol andpolyethylene glycol and their derivatives, alginates such as sodiumalginate or crosslinked alginate gum, polycaprolactone, polyanhydride,pectin, gelatin, crosslinked proteins peptides and polysaccharides, andthe like.

Hydrogels of the present invention can be formed according to any methodas is generally known in the art. For instance, the hydrogel canself-assemble upon contact of the various components or upon contact inconjunction with the presence of particular environmental conditions(such as temperature or pH). Alternatively, assembly can be inducedaccording to any known method following combination of the components.For example, step-wise or chain polymerization of multifunctionalmonomers, oligomers, or macromers can be induced viaphotopolymerization, temperature dependent polymerization, and/orchemically activated polymerization. Optionally, the hydrogel can bepolymerized in the presence of an initiator. For example, in oneembodiment, the hydrogel can be photopolymerized in the presence of asuitable initiator such as Irgacure® or Darocur® photoinitiatorsavailable from Ciba Specialty Chemicals. In another embodiment, acationic initiator can be present. For example, a polyvalent elementalcation such as Ca²⁺, Mg²⁺, Al³⁺, La³⁺, or Mn²⁺ can be used. In anotherembodiment, a polycationic polypeptide such as polylysine orpolyarginine can be utilized as an initiator.

The components of the hydrogel delivery vehicle can also be designed soas to provide a self-assembling delivery vehicle. For example, ahydrogel precursor can be administered to a subject, and the hydrogelmatrix can self-assemble at physiological conditions followingadministration of the precursor. For instance, the hydrogel precursorcan include self-assembling biopolymers such as collagens, laminins,pro-elastin peptides, and the like. Optionally, a self-assemblinghydrogel precursor can include synthetic polymers that can arraythemselves according to domains, as is generally known in the art. Forexample, hydrophilic, relatively charge-neutral synthetic polypeptidessuch as polyglycine or polylysine can be modified to function in thiscapacity. Polypeptides can be crosslinked by using carboxy-activatingcrosslinking agents such as water-soluble carbodiimides. Suchcross-linking agents can be used to attach self-assembling proteins orother self-assembling macromolecules to the polypeptides. One example ofthis approach includes formation of a carbodiimide linkage of collagenor laminin with polylysine. Other hydroxylated entities can be linked ina similar manner. For example, in one embodiment, polyvinyl alcohol canbe linked with polypeptides using an epoxy-activation approach orcrosslinked via polymerizable methacrylate groups along its side chains,as is known in the art.

In another embodiment, a self-assembling hydrogel can be generated byuse of precursors that have been derivatized to contain favorablyreactive groups. For example, a hydrogel of this type could be assembledusing a first precursor derivatized with a particular reactive moietyand a second precursor derivatized with or comprising a second moietythat can preferentially react with the first moiety on the firstprecursor. Likewise, other such hydrogels could be generated using suchreactive pairs wherein the two moieties that react to form the bond areeach conjugated to the same or a different type of polymer. For example,the pairs can be antibody-antigen pairs or avidin-biotin (e.g.streptavidin-biotin).

In other embodiments a hydrogel delivery vehicle need not be aself-assembling matrix. For example, in other embodiments a hydrogelmatrix for use in vivo can be administered to a patient according to asuitable administration method (e.g., percutaneously) following assemblyof the hydrogel. In other embodiments of the invention, the disclosedsystems can be utilized in ex vivo applications, for example in tissueengineering applications, and as such, the carrier matrix of theinvention need not be a self-assembling matrix.

The delivery vehicles of the present invention can include a combinationof one or more delivery vehicles. For instance, a hydrogel deliveryvehicle can be combined with a patch, a stent, a perforated balloon, avascular graft, or any other suitable device, for delivery of thedisclosed agents to connective tissue.

Delivery vehicles of the invention can also include vascular grafts. Forexample, an allograft, xenograft or autologous graft can be associatedwith a phenolic compound as herein described prior to implantation. Forexample, the vascular graft can be coated with a phenolic compound or acomposition including a phenolic compound as herein described. Inanother embodiment, a vascular graft can be associated with a hydrogeldelivery vehicle or a non-hydrogel polymeric delivery vehicle such asthose described above that has in turn been loaded with the phenoliccompound. During implantation, the vascular graft can be located inassociation with the targeted connective tissue, and thus serve todeliver the phenolic compound to the tissue.

Reference now will be made to exemplary embodiments of the invention setforth below. Each example is provided by way of explanation of theinvention, not as a limitation of the invention. In fact, it will beapparent to those skilled in the art that various modifications andvariations may be made of this invention without departing from thescope or spirit of the invention.

EXAMPLES

Materials utilized in all examples were obtained as follows: Tannicacid, glutaraldehyde (50% solution), and other high purity chemicalswere obtained from Sigma-Aldrich (St. Louis, Mo.). Diethyl ether wasobtained from Acros Organics (Morris Plains, N.J.). Ethyl acetate wasacquired from EM Science (Gibbstown, N.J.). High purity porcinepancreatic elastase (135 U/mg) was purchased from Elastin ProductsCompany, Inc. (Owensville, Mo.). Dulbecco's modified eagle media (DMEM)and fetal bovine serum (FBS) were obtained from Cellgro (Herndon, Va.).Penicillin-streptomycin for cell culture was obtained from Invitrogen(Carlsban, Calif.). CellTiter 96® AQ_(ueous) One Solution Reagent (forMTS) was from Promega (Madison, Wis.). The LIVE/DEAD®Viability/Cytoxicity Kit was obtained from Molecular Probes (Eugene,Oreg.).

Ascending porcine aorta (supravalvular segments, approximately 3 cm inlength) were collected fresh from USDA-approved slaughterhouses andtransported on ice. Aorta was cleaned of fat and extraneous tissue,rinsed exhaustively in cold physiological saline, and cut into variousshapes appropriate for each example.

Example 1

Fresh porcine aorta was cut into approximately 4 mm×4 mm squares andtreated with 0.3% tannic acid (TA) (1.76 mM) in 50 mM HEPES (Na₂HPO₄)buffered saline at pH 5.5 for 4 days at room temperature. A controlsample was treated in mM Hepes buffered saline at pH5.5. Samples werethen rinsed three times (1 hour each) in 100 mL ddH₂O at roomtemperature and lyophilized to record dry weight. Samples (about 15-25mg dry weight), both control and TA treated, were incubated with 1.0 mLpure elastase (20 units/ml, in 100 mM Tris, 1 mM CaCl₂, 0.02% NaN₃, pH7.8 buffer) at 37° C. for 48 hours with orbital shaking at 650 rpm.Samples were then centrifuged (10000 rpm, 10 min. 4° C.) andsupernatants retained to assess enzyme activity. Tissue samples wereagain rinsed three times (1 hour each) in 100 mL ddH₂O at roomtemperature and lyophilized to record dry weight after elastase, and thepercent of digested tissue was calculated. Elastin degradation wasassessed by measuring mass loss (dry weight before and after elastasecontact) and by preparation of histological slides stained with anelastin-specific stain (Voerhoff van Gieson stain).

Upon assessment, the control samples were found to have lost almost 80%of the mass (average, 79.74%, n=6), while the TA pre-treated sampleslost only 4-5% of the mass (average 4.33%, n=6). Histology showed thatelastin fibers were completely digested by elastase in the controlsamples, while in TA pre-treated samples, elastin fibers were fullypreserved, as can be seen in FIGS. 4A-4D, which illustrate the resultsof the histology study. FIGS. 4A-4D show aortic tissue sections stainedwith Voerhoff van Gieson stain, which reveals elastin fibers as darkribbons. In particular, FIGS. 4A and 4B show the fresh control aortabefore and after the procedure, respectively, and FIGS. 4C and 4Dillustrate the TA pre-treated samples before and after the procedure,respectively. As can be seen, elastin was completely digested by theelastase in the control aorta (FIG. 4B), as compared to fullpreservation of the elastin the TA pre-treated aorta (FIG. 4D). Thearrows in the Figures point to locales including the preserved elastin(FIGS. 4A, 4C, and 4D) and the digested elastin (FIG. 4B).

Example 2

Fresh porcine aorta was cut across the diameter into continuous ringsapproximately 1 cm in height as illustrated in FIG. 5A and treated withglutaraldehyde (Glut treatment). For this treatment, aortic ring sampleswere first fixed with 0.6% glutaraldehyde in 50 mM Hepes bufferedsaline, pH 7.4, overnight at room temperatures, followed by 0.2%glutaraldehyde in the same buffer for 7 days at room temperatures.

A portion of the Glut-treated rings were then treated with 0.3%pentagalloylglucose (PGG). PGG was first prepared from TA as outlined byHagerman, et al. (Hagerman A. E., Zhao Y., and Johanson J., “Methods fordetermination of condensed and hydrolyzable tannins,” Shahadi, F., Ed.,Antinutrients and Phytochemicals in Foods, Washington, D.C., AmericanChemical Society, p. 209-222 (1997)). Specifically, TA was methanolyzedusing a solution of methanol and acetate buffer. After methanolysis, themethanol was removed by rotary evaporation and replaced with ddH₂O. Aseries of separate extractions involving diethyl ether and ethylacetate, along with rinses of dilute methanol and ddH₂O was performed topurify the PGG. The resulting precipitate was centrifuged andlyophilized to form a solid. The purity of PGG was confirmed by MALDImass spectroscopy and NMR. A portion of the Glut-treated tissue was thentreated with PGG in the same buffer for 4 days (Glut/PGG treatment,marked as PGG on FIG. 5).

Another Glut-treated sample was treated with TA according to the sameprocedure (Glut/TA treatment, marked as TA on FIG. 5), and a controlsample was treated in only the buffer (marked as Fresh on FIG. 5).

Samples were then tested for ability to recoil using opening anglemeasurements. Specifically, each ring was placed in a dish of water(enough to completely cover the tissue) with the cross section of theaorta facing upward, allowing free movement of the sample. The ringswere then cut once in the radial direction, allowed to relax and openfor 15 minutes, and then digitally photographed. These digitalphotographs were imported into Adobe® Photoshop® 7.0 and lines werecreated for each sample between the tip of the cut edges and themidpoint of the luminal wall opposite the location of the radial cut, asshown in the Glut sample photograph in FIG. 5C. Using these lines, theopening angles of the aortic rings were calculated. Results areillustrated in FIG. 5B and Table 1, below, including standard error ofthe mean (SEM) values.

To evaluate mechanical properties, aorta was cut to form dumbbell shapesapproximately 40 mm in length, approximately 10 mm wide at each end, andapproximately 5 mm wide at the center, with the long axis of thedumbbell shape being parallel to the circumferential direction of theaorta. Tensile properties of the samples were tested using standardstress/strain analysis on a Synergie 100 testing apparatus (MTS SystemsCorporation, Eden Prairie, Minn.) operated at a constant uniaxialvelocity of 0.2 mm/sec using a 10-Newton load cell to determine elasticmodulus of the samples. Elastic modulus was calculated as the slope ofthe stress-strain curve between 0 and 5% strain. Results are shown belowin Table 1.

TABLE 1 Mean opening angle SEM Mean Elastic SEM Pre-treatment (degrees)(n = 5) Modulus (kPa) (n = 4) Control 177.50 11.0 0.16 0.01 Glut 35.93.3 1.28 0.04 Glut/TA 11.8 0.7 4.85 0.30 Glut/PGG 5.9 0.7 4.61 0.40

As can be seen, Glut/TA and Glut/PGG treated rings exhibited muchsmaller opening angles, indicating that tannic acid interacted with theelastin component and did not fully allow recoil to occur. Similarly,the calculated elastic modulus was increased following treatment withtannic acid and PGG. These results suggest that the tannins bindstrongly to the elastic components of the tissue and in doing so stiffenand strengthen the tissue.

Example 3

Fresh porcine aorta was treated with Glut, Glut/TA, or Glut/PGG, asdescribed above. After thorough rinsing (3×, 1 hr. each, 100 mL ddH₂O),samples were orbitally shaken at 120 rpm 37° C. for 14 days in aPBS/azide buffer, pH7.4, for extraction of soluble, cytotoxic compounds(50 mL per 6 tissues samples, each 4 mm×4 mm).

Rat skin fibroblasts were seeded onto 24-well plates (50,000 cells/well)in 1 mL DMEM supplemented with 10% FBS and 1% penicillin-streptomycin.Cells were maintained in a humidified incubator at 37° C. with 10% CO₂.After 24 hours, the media was removed and replaced with one of thePBS/azide solutions diluted 10-fold in fresh media. As control, a samplewas held in buffer alone (negative control) and also in a 70% ethanolsolution (positive control). Effectively, 100 μL of the ‘test solution’was added to 900 μL of media for each well (except in the case ofethanol, in which no media was added). The cells were incubated withthese solutions for 2 hours and then rinsed with 1 mL PBS. Viability ofthe cells was then assessed using a qualitative LIVE/DEAD® stainingfluorescent stain. In addition, a quantitative measurement, MTS, wasused to measure the activity of cell enzymes. Specifically, the reagentwas diluted 5-fold in media, then applied to the cells for 90 minutes. Aportion of the resulting solutions (100 μper well) was transferred to a96-well plate and read at an absorbance of 490 nm. Samples were blankedto a well containing media and MTS reagent with no cells.

Micrographs of cells introduced to LIVE/DEAD® were taken 45 minutesafter exposure. Results are shown in FIGS. 6A-6E, in which living cellsappear as light spots on the dark background, and dead cells appear asdarker gray spots. As can be seen with reference to the Figures, cellsexposed to extracts from Glut (FIG. 6B) and Glut/PGG-treated (FIG. 6A)tissue remained viable after 2 hours of exposure. In addition, itappeared that the small concentration of azide had little or no effecton cytotoxicity in this time frame, as suggested by the presence of livecells in the PBS/azide group (FIG. 6D). However, cells that wereintroduced to extracts from Glut/TA-treated aorta experienced a notableamount of cell death (about 40%, FIG. 6C). As expected, the EtOHnegative control left essentially no viable cells (FIG. 6E).

Quantitative results from the MTS assay are given below in Table 2.These results suggest that phenolic tannins are generally not cytotoxic,but PGG appears to be less cytotoxic than TA.

TABLE 2 Viability Viability SEM Pre-treatment (live/dead assay) (MTSassay) (n = 3) Glut 100% 1.04 Glut/PGG 100% 0.94 0.03 Glut/TA 60% 0.530.06 EtOH 0% 0.03 0.02 PBS/azide 100% 1.04 0.00

Example 4

Ascending porcine aorta was collected fresh at a slaughterhouse andtransported to the laboratory on ice. After cleaning fat and adherenttissues, aorta was cut into 2×4 mm strips and thoroughly rinsed in coldsaline. Aortic elastin was purified by a sequence of extractions.Specifically, aortic strips were suspended in 100 mM sodium hydroxide(60 strips in 100 mL) and incubated for 14 hours at 37° C. withcollagenase (0.5 units/mg wet tissue) in 50 mM Tris buffer, 10 mMcalcium chloride, pH 8.0. The collagenase preparation was pre-adsorbedwith elastin fibers to remove residual elastolytic activities. The finaldigestion step completely removed residual collagen, leaving pure aorticelastin.

The pure aortic elastin strips were placed in 2 mL microfuge tubes andsuspended in 1.5 mL of an 8 mg % TA solution prepared in a 50 mMdisodium hydrogen phosphate, 0.9% sodium chloride buffer, pH 5.5. Asecond group consisted of pure aortic elastin suspended in a solutioncontaining 8 mg % TA and 16 mg % Glut in the same buffer. As controls,TA solution and the mixture of TA and Glut were incubated withoutelastin samples. Samples were incubated at room temperature and atdefined time intervals (0, 20, 40, 60, 120, 360 minutes and 24 hours),samples were retrieved and analyzed for TA content in solution.Specifically, samples were mixed with a tungstate/phosphomolybdatereagent followed by addition of a saturated sodium carbonate solutionand ddH2O. After 10 minutes at room temperature, OD at 760 nm wasmeasured in a microplate spectrophotometer. Standard curves wereconstructed with TA in the range of 0-8 mg % and with TA (0-8 mg %) inmixtures with Glut (16 mg %). Glut interference with the TA colorreaction was minimal (no statistical differences found for all datapoints). Finally, the elastin strops were rinsed in ddH2O andlyophilized. The amount of TA bound to pure aortic elastin wascalculated from the differences between initial TA concentration insolution and concentration of TA in solution after incubation withelastin strops and were expresses as micrograms of TA bound by 1 mg dryelastin. All samples were assayed in triplicate.

Results are graphically illustrated in FIG. 7. As can be seen withreference to the figure, levels of TA in control solutions incubatedwithout elastin remained constant throughout the study, indicating thatthe solutions were stable. However, within 1 hour of incubation withelastin, the amount of TA in solution decreased by about 50%, decreasingto 10% after 6 hours, and to less than 3.5% of initial TA at 24 hours,clearly indicating binding of TA to elastin. The kinetics shows a rapidbinding within the first 6 hours, leveling off at about 3 mg of TA/mgdry elastin. The binding rate of TA from mixtures of TA and Glut wasfound to be higher that that of TA alone, suggesting that Glut mayfacilitate or accelerate binding of TA to elastin.

Example 5

In vivo use of the disclosed agents was examined. The aneurysm modelused was based on the perivascular application of a high concentrationcalcium chloride (CaCl₂) solution, a method originally used to induceaneurysms in rabbit carotid arteries (see, Gertz S D, Kurgan A,Eisenberg D. J Clin Invest 1988; 81(3):649-656.) and has more recentlybeen used on abdominal aorta of rodents. (See, e.g., Freestone T., etal., Arterioscler Thromb Vasc Biol 1995; 15(8):1145-1151. Freestone T.,et al., Arterioscler Thromb Vasc Biol 1997; 17(1):10-17. Tambiah J., etal. Br J Surg 2001; 88(7):935-940.) This model results in a localizedmild insult to the arterial tissue. In most studies, a significantincrease in aortic diameter (indicative of aneurysm formation) wasobserved after 3 to 6 weeks post-injury. For this example, theCaCl₂-based chemical injury was performed on rat aorta.

Adult male Sprague-Dawley rats were placed under general anesthesia (2%to 3% isoflurane), allowing for a midline incision along the abdomen inorder to expose the infrarenal abdominal aorta. Once exposed, theabdominal aorta was treated with 0.03% PGG solution in saline for 15minutes using a strip (1.5 cm×0.5 cm) of presoaked 8-ply sterile cottongauze. The area was then briefly rinsed with saline, allowing inductionof chemical injury (0.5 M CaCl₂, delivered with gauze for 15 minutes).As controls, rat aortas were treated with physiological saline for 15minutes, rinsed, and then subjected to calcium chloride. After treatingwith CaCl₂, the gauze was removed and the abdominal cavity was closedwith subcutaneous suture, followed by surgical staples. The rats wereanesthetized after 28 days, at which point the abdominal aorta was againexposed. Digital photographs (before PGG application at day 0, as wellas after 28 days) were used to measure percent increases in diameterafter the 28 day period for each rat. Aortic tissue was collected foranalysis.

FIG. 8 graphically illustrates the absolute change in diameter of theaortic segments (n=12 per group). As can be seen, in the control aortas,the increase in diameter of the aortic segments was about 530 μm. In thePGG-treated segments, in contrast, the increase was about 100 μm, andthe differences were found to be statistically significant. FIG. 9 insetphotographs show an example of a control aorta, pre-injury, a controlaorta 28 days post-injury, and a PGG-treated aorta, 28 days post-injury.FIG. 9 graphically shows the results according to the percentage changein diameter of the segments—greater than 40% for the control, and lessthan 10% for the PGG-treated aortas.

Following superficial examination of the aortic segments, the segmentswere analyzed via desmosine analysis for elastin content. According tothe analysis protocol, higher desmosine content implies lower elastindegradation. The results are illustrated in FIG. 10. As can be seen withreference to the Figure, the control included about 750 pmoldesmosine/mg protein, while the PTT-treated aortas included about 1350pmol desmosine/mg protein. FIGS. 10B and 10C illustrate a control aortaand a treated aorta, respectively, following Verhoeff van Giessonstaining, which stains elastin black. As can be seen the control aortahas very little elastin, while the treated aorta has retained elastin(shown by the black staining of the elastic fibers in the figures).

Vessels were also analyzed to determine the long-term binding of thetannins with the tissue. Tannins were extracted from explanted aortaimmediately after treatment (labeled PGG in situ on FIG. 11) andmeasured levels were compared to those obtained from aorta explanted 28days following PGG treatment (labeled PGG in vivo on FIG. 11). As can beseen, while the level of tannins found in the tissue dropped somewhatover time, from about 1.8 μg PGG/mg dry tissue to about 1.3 μg PGG/mgdry tissue, a larger percentage of the tannins remained bound to thetissue in vivo for the extended period.

It will be appreciated that the foregoing examples, given for purposesof illustration, are not to be construed as limiting the scope of thisinvention. Although only a few exemplary embodiments of this inventionhave been described in detail above, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention which isdefined in the following claims and all equivalents thereto. Further, itis recognized that many embodiments can be conceived that do not achieveall of the advantages of some embodiments, yet the absence of aparticular advantage shall not be construed to necessarily mean thatsuch an embodiment is outside the scope of the present invention.

1. A leaky balloon for repair of aneurysm of a blood vessel in a livingsubject comprising a balloon element and a therapeutic compositioncontained in the balloon element, wherein the balloon element comprisesa membrane that allows the therapeutic composition to leak from theballoon element to access the aneurysm, wherein the therapeuticcomposition is substantially free of unbound gallic acid and has a pHbetween about 4 and about 9, and wherein the therapeutic compositioncomprises a phenolic compound having a plurality of phenolic groups atan effective concentration that crosslinks elastin in the blood vesselin vivo to stabilize the blood vessel.
 2. The leaky balloon of claim 1wherein the phenolic compound is a flavonoid or a flavonoid derivative,a flavolignan or a flavolignan derivative, a phenolic rhizome or aphenolic rhizome derivative, a flavan-3-ol or a flavan-3-ol derivative,a tannin or a tannin derivative, an ellagic acid or an ellagic acidderivative, a procyanidin or a procyanidin derivative,proanthrocyanidin, anthocyanins, quercetin, (+)-catechin,(−)-epicatechin, nobotanin, epigallocatechin gallate, gallotannins,hydroxytyrosol (3,4-dihydroxy phenylethanol), oleuropein,prodelphinidin, a combination thereof, or a pharmaceutically acceptablesalt thereof.
 3. The leaky balloon of claim 1 wherein the phenoliccompound comprises tannic acid or an analog of tannic acid,pentagalloylglucose or an analog of pentagalloylglucose, a combinationthereof, or a pharmaceutically acceptable salt thereof.
 4. The leakyballoon of claim 1 wherein the therapeutic composition comprises about0.0001 w/v % and about 10 w/v % of the phenolic compound.
 5. The leakyballoon of claim 1 wherein the phenolic compound stabilizes the elastinof the blood vessel through cross linking with the elastin protein. 6.The leaky balloon of claim 1 wherein the therapeutic composition furthercomprises a drug delivery vehicle comprising a sustained release drugdelivery vehicle, a hydrogel, a perivascular drug delivery vehicle, anendovascular drug delivery vehicle, or a combination thereof.
 7. Theleaky balloon of claim 6 wherein the drug delivery vehicle comprises ahydrogel comprising glycosaminoglycans, polysaccharides, proteins,dextran, hyaluronic acid, chitin, heparin, collagen, elastin, keratin,albumin, polymers and copolymers of lactic acid, glycolic acid,carboxymethyl cellulose, polyacrylates, polymethacrylates, epoxides,silicones, polyols such as polypropylene glycol, polyvinyl alcohol andpolyethylene glycol and their derivatives, alginates such as sodiumalginate or crosslinked alginate gum, polycaprolactone, polyanhydride,pectin, gelatin, crosslinked proteins peptides and polysaccharides, or acombination thereof.
 8. The leaky balloon of claim 6 wherein thetherapeutic composition is encapsulated in a drug delivery vehicle. 9.The leaky balloon of claim 6 wherein the drug delivery vehicle isbiodegradable.
 10. A method of repairing aneurysm in a blood vessel of aliving subject using the leaky balloon of claim 1, the methodcomprising, deploying the leaky balloon at the aneurysm in the bloodvessel, and eluting the therapeutic composition from the leaky balloonto stabilize the blood vessel at the aneurysm.
 11. The method of claim10 further comprising treating the blood vessel before, during, or afterthe deployment of the leaky balloon with glutaraldehyde.
 12. The methodof claim 10 wherein the aneurysm is treated with the leaky balloonmultiple times.
 13. The method of claim 10 wherein the leaky balloon isdeployed in the aneurysm for sufficient period of time to allow thephenolic compound to cross link the elastin of the blood vessel.